WO2007100727A2 - Des champs électriques pulsés en nanoseconde causent l'autodestruction de mélanomes - Google Patents

Des champs électriques pulsés en nanoseconde causent l'autodestruction de mélanomes Download PDF

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WO2007100727A2
WO2007100727A2 PCT/US2007/004844 US2007004844W WO2007100727A2 WO 2007100727 A2 WO2007100727 A2 WO 2007100727A2 US 2007004844 W US2007004844 W US 2007004844W WO 2007100727 A2 WO2007100727 A2 WO 2007100727A2
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cells
tumor
nspef
electric field
pulse
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WO2007100727A3 (fr
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Richard Nuccitelli
Stephen J. Beebe
Karl H. Schoenbach
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Eastern Virginia Medical School
Old Dominion University
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Eastern Virginia Medical School
Old Dominion University
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Priority to US12/280,280 priority Critical patent/US9168373B2/en
Priority to JP2008556458A priority patent/JP5329978B2/ja
Priority to AU2007221182A priority patent/AU2007221182B2/en
Priority to CA2643210A priority patent/CA2643210C/fr
Publication of WO2007100727A2 publication Critical patent/WO2007100727A2/fr
Publication of WO2007100727A3 publication Critical patent/WO2007100727A3/fr
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Priority to US14/869,286 priority patent/US9943684B2/en
Priority to US15/922,758 priority patent/US10905874B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
    • A61N1/205—Applying electric currents by contact electrodes continuous direct currents for promoting a biological process
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • A61N1/36002—Cancer treatment, e.g. tumour

Definitions

  • nsPEF nanosecond pulsed electric fields
  • nsPEF nanosecond pulsed electric fields
  • the interior charges will not have sufficient time to redistribute to counteract the imposed field and it will penetrate into the cell and charge every organelle membrane for a duration which is dependent on both the charging time constant of the cell's plasma membrane as well as that of the organelle membrane (K.H.Schoenbach, R.P.Joshi, J.F.Kolb, N.Chen, M.Stacey, P.F.BIackmore,E.S.Buescher, S.J.Beebe (2004) Proc. IEEE. 92:1122- 1137).
  • a second critical nsPEF parameter is the amplitude of the pulse. Both the force exerted on charges and the electroporation of lipid membranes depend on the strength of the electric field. When the electric field across a cellular membrane exceeds about 1 volt (2 kV/cm for a cell 10 ⁇ m in diameter), water-filled pores form in the membrane's lipid bilayer and the size and lifetime of these pores are dependent on the strength and duration of the electric field pulse.
  • heating is proportional to pulse duration and the square of the field strength, the much shorter pulses in the nanosecond range can have a higher field strength while delivering the same low level of thermal energy to the tissue.
  • a 20-fold higher field strength of 40 kV/cm can be employed to generate structural changes in the plasma membrane that result in a smaller electrical barrier as well as higher voltage gradients across cellular organelles for die duration of the pulse (Q.Hu, S.Viswanadham, R.PJoshi, K.H.Schoenbach, SJ.Beebe, P.F.Blackmore (2005) Phys.Rev.E Stat.Nonlin.So ⁇ .Matter Phys.7 ⁇ :031914-l-0319l4-9).
  • a typical tumor cell nucleus measuring 10 ⁇ m in diameter will experience a voltage gradient of roughly 40 V across its diameter during each pulse. This electric field is large enough to cause electrodeformation (R.PJoshi, Q.Hu, K.H.Schoenbach, H.P.Hjalmarson (2002) Phys.Rev.E Stat.Nonlin.Soft.Matter Phys. 65:021913).
  • This elongation in DNA electrophoresis tracks is normally interpreted to indicate fragmentation of the DNA into smaller pieces that is associated with apoptotic cell death.
  • An indication of changes in the DNA following nsPEF treatment comes from images of the nucleus labeled with acridine orange, a vital fluorescent dye that intercalates into DNA and RNA, Chen et al. (N.Chen, K.H.Schoenbach, J.F.Kolb, S.RJames, A.L.Garner, J.Yang, R.PJoshi, SJ.Beebe (2004) Biochem.Biophys.Res.Commun. 317:421-427).
  • a single 10 ns pulse of 26 kV/cm caused a dramatic decrease in fluorescence intensity in the nucleus evident as early as 5 min after the pulse. This change could be due to an outflow of DNA or to conformational changes in the DNA.
  • the ability to selectively modify specific cells in ways that lead to apoptosis could provide a new method for the selective destruction of undesired tissue (e.g., cancer cells, fat cells or cartilage cells) while minimizing side effects on surrounding tissue.
  • An electrical method of treatment that results, not only in tumor growth inhibition, but in complete tumor regression, without hyperthermia, drugs, or significant side effects, would be a great advancement in the field of cancer therapy and other in situ therapies.
  • One or more aspects of the invention provide a method for selectively initiating apoptosis in target cells in a tissue.
  • the method comprises applying at least one nsPEF to said tissue.
  • the at least one nsPEF has a pulse duration of at least about 10 nanoseconds and no more than about 1 microsecond and an electric field pulse strength of at least about 10 kV/cm and no more than about 350 kV/cm.
  • the method is carried out in situ.
  • At least one nsPEF has a pulse duration of about 300 nanoseconds and an electric field pulse strength of at least about 20 kV/cm and no more than about 40 kV/cm.
  • At least 100 nsPEFs are applied to said tissue.
  • at least 300 nsPEFs are applied to the tissue.
  • at least 400 nsPEFS are applied to the tissue.
  • the method of treatment of at least one nsPEF is repeated.
  • the target cells are fat cells. In one or more aspects of the invention, the target cells are bone cells. In one or more aspects of the invention, the target cells are vascular cells. In one or more aspects of the invention, the target cells are muscle cells. In one or more aspects of the invention, the target cells are cartilage cells. In one or more aspects of the invention, the target cells are stem cells. In one or more aspects of the invention, the target cells are a combination of the above cells.
  • a method for inhibiting blood flow in a tissue comprises applying at least one nsPEF to said tissue.
  • the at least one nsPEF has a pulse duration of at least about 10 nanoseconds and no more than about 1 microsecond and an electric field pulse strength of at least about 10 kV/cm and no more than about 350 kV/cm. In one or more embodiments of the invention, the method is carried out in situ. [0013]
  • the invention also provides a method for inducing tumor regression. The method comprises applying at least one nsPEF to said tumor.
  • the at least one nsPEF has a pulse duration of at least about 10 nanoseconds and no more than about 1 microsecond and an electric field pulse strength of at least about 10 kV/cm and no more than about 350 kV/cm. In one or more embodiments of the invention, the method is carried out in situ.
  • Figure 1 depicts the pulse generator used in these experiments.
  • A 300 ns pulse- forming network in Blumlein configuration.
  • B Typical voltage and current pulse generated across a tumor.
  • Figure 2 depicts the needle array electrode and electric field pattern.
  • A Photograph of 5 needle array used for the first experiments.
  • B 3-D plot of the electric field generated when 8 kV is placed on the center electrode and the outer four electrodes are held at ground.
  • Figure 3 shows the typical response of skin and melanoma to one or two applications of 100 pulses using a 5-needle array electrode on mouse #56. Each matched pair of photos represents an in situ transillumination of the skin on the left and a surface view on the right. Numbers on the far left indicate the number of days after pulsing at which all three matched pairs to the right were photographed.
  • A-F The typical response of normal skin to 100 pulses (300 ns long, 20 kV/cm, 0.5 Hz) delivered on day 0. Small superficial erosion in B grows in C-E and indicates loss of some or all epidermis.
  • H-M The electrode array was inserted into this tumor on day 0 but no pulses were delivered.
  • O-T 100 pulses (300 ns long, 20 kV/cm) were delivered at 0.5 Hz on day 0 and day 1. Necrosis evident on day two becomes more intense over time. Scale bars A-T: 1 mm and all photos in a given row are at the same magnification.
  • Figure 4 provides a summary of the size changes in a total of 23 melanomas after the indicated treatments using the 5-needle array. For each day the tumor area was measured from the transillumination image and divided by that measured on day zero to give the normalized area. The average response of two to three tumors from different animals is plotted on a logarithmic scale and the error bars represent the S.E.M. Pulses were applied at a frequency of 0.5 Hz. (A-B) 4 kV was applied between center and outer needles spaced 4 mm apart to give an average field of 10 kV/cm. C-E: 8 kV was applied between the center and outer needles to give an average field of 20 kV/cm,
  • Figure 5 depicts a typical response of a melanoma to three applications of 100 pulses (300 ns, 40 kV/cm, 0.5 Hz) 30 minutes apart on day 0 followed by a single application on day 4 using a 5 mm diameter parallel plate electrode on mouse #102. Collection of 7 matched sets of images of the same tumor all taken on the day indicated in the lower left corner of the transillumination image.
  • Column A Transillumination image.
  • Column B Surface view.
  • Column C Ultrasound slice at center of tumor;
  • Column D 3-D reconstruction made from 100 serial ultrasound slices through tumor. Magnification is constant for each column and scale bar at top of each column represents 1 mm.
  • FIG. 6 shows a photograph of SKH-I hairless mouse being treated with parallel plate electrode under isoflurane inhalation anesthesia. Inset: Close-up of one of the plates of parallel plate electrode showing it recessed by 0.5 mm to allow a space for a conductive agar gel to be placed on it.
  • (B) Mean change in normalized area of the transillumination image of 6 tumors from 3 mice treated with parallel plate electrodes using the same 4x100 pulse applications (3x100 on day 0 and 1x100 on day 4). 40-80 kV/cm, 300 ns pulses at 0.5 Hz. Error bars indicate the S.E.M.
  • Figure 7 shows complete regression of melanoma evident by 65 days after the first treatment. 100 pulses of 300 ns and 40 kV/cm were applied on days 0, 1, 2 and 21, 22, 23. Each pair of photos were taken on the day indicated at the left; transillumination on left and surface view on right. The scale bar in upper left represents 1 mm and is the same for all images.
  • Figure 8 depicts the measurement of the temperature within a melanoma during nsPEF application.
  • A Micrograph of a thermocouple made by fusing a copper wire with one made from constantine.
  • B Temperature record from a thermocouple positioned inside of a melanoma during pulse application. Red dots indicate the time that each pulse was applied.
  • Figure 9 depicts targets and mechanisms of nsPEF effects.
  • A-D 7 ⁇ m thick paraffin sections of control and treated melanomas fixed at the indicated time after treatment with 100 pulses (300 ns, 40 kV/cm, 0.5 Hz) stained with hematoxylin and eosin.
  • Figure 10 shows the blood flow in melanoma before and after nsPEF application.
  • A 3-D reconstruction of volume of melanoma
  • B Power Doppler reconstruction of blood flow before field application.
  • C 3-D reconstruction of volume of same melanoma shown in
  • Doppler reconstruction of blood flow in the same tumor shown in B generated about 15 minutes after 100 pulses (300 ns, 40 kV/cm, 0.5 Hz)
  • Figure 11 shows transillumination views of one control and three treated tumors at the day indicated at the top of each column. Photo in day 0 was taken just before the first nsPEF application. A second application of 300 pulses occurred on day 15. No other treatments were needed and these animals remain tumor-free to date.
  • Figure 12 shows a UV-induced melanoma in a HGF/SF transgenic mouse that was treated on day 0 with 300 pulses 300 ns long and 40 kV/cm in amplitude.
  • 3D reconstruction of serial section; ultrasound images (top row) and surface micrographs (bottom row) indicate that the tumor shrinks rapidly over the 19-day period studied to date.
  • Figure 13 shows the computed electrical field distribution (in arbitrary units for a two-needle electrode configuration system in a linear array). The series of photographs on the right shows the temporal development of the tumor.
  • Biological cells consist of cytoplasm surrounded by a membrane.
  • the cytoplasm is conducting, while the membrane, which is made up of a lipid bilayer, can be considered a dielectric.
  • the application of electric fields to biological cells causes buildup of electrical charge at the cell membrane, and consequently a change in voltage across the membrane.
  • the transmembrane voltage under equilibrium condition is approximately 70 mV.
  • the amplitude of these electric fields must be such that it generates a potential difference ("V m ") at least on the same order as the resting potential.
  • V m potential difference
  • E V m /fa (1)
  • a is the radius of the cell and f is a form factor which depends on the shape of the cell.
  • the external electric field required to generate a voltage of the same amplitude as the resting potential across the membrane is on the order of 100 V/cm.
  • nsPEF or “nanosecond pulsed electric field” as used herein is defined as an electric pulse in the nanosecond range (about 100 picoseconds to about 1 microsecond) with electric field intensities from about 10 kV/cm to about 350 kV/cm.
  • any apparatus equipped with a pulse generator that can deliver short electrical pulses of pulse duration of at least about 100 picoseconds and no more than about 1 microsecond, and of electric field strength of at least about 10 kV/cm and no more than about 350 kV/cm may be used.
  • the pulse generator can deliver short electrical pulses of pulse duration of at least about 100 picoseconds and no more than about 1 microsecond, and of electric field strength of at least about 10kV/cm and no more than about 40 kV/cm. In another aspect of the invention, the pulse generator can deliver short electrical pulses of pulse duration of at least about 100 picoseconds and no more than about 1 microsecond, and of electric field strength of at least about 20 kV/cm. and no more than about 125 kV/cm.
  • the pulse generator can deliver short electrical pulses of pulse duration of at least about 10 nanoseconds and no more than about 300 nanoseconds, and of electric field strength of at least about 20 kV/cm and no more than about 45 kV/cm. In another aspect of the invention, the pulse generator can deliver short electrical pulses of pulse duration of at least about 10 nanoseconds and no more than about 350 nanoseconds, and of electric field strength of at least about 20kV/cm and no more than about 125 kV/cm. In another aspect of the invention, the pulse generator can deliver short electrical pulses of pulse duration of about 10 nanoseconds and an electric field strength of about 125 kV/cm. In another aspect of the invention, the pulse generator can deliver short electrical pulses of pulse duration of about 300 nanoseconds and an electric field strength of about 40 kV/cm.
  • the apparatus for delivery of nsPEFs is also equipped with a high voltage power supply and with a means for directing the nsPEFs to the target cells.
  • the target cells are in situ, and any suitable means for directing the nsPEFs to the in situ target cells may be employed.
  • Suitable means for directing the nsPEFs will preferably allow high voltage, short duration electrical pulses in the nanosecond range, for example, within tissues. Examples include an electrode system, such as plate electrodes, needles or needle arrays.
  • the nsPEFs are applied directly to cells present as part of a tissue.
  • nsPEF pulses of the present invention can be administered to the cells by means of a pulse generator, such as the generator previously described in U.S. Patent No. 6,326,177 and Beebe et al. FASEBJ. 17, 1493-1495 (2003).
  • a pulse generator such as the generator previously described in U.S. Patent No. 6,326,177 and Beebe et al. FASEBJ. 17, 1493-1495 (2003).
  • the application of these high frequency intracellular effects had been limited due to the difficulty of generating large intracellular electric fields on a time scale that is comparable to or even less than the charging time of the surface.
  • the present inventors developed technology for generating high voltage, short duration electrical pulses that make it possible to produce electric pulses in the nanosecond range with voltage amplitudes adequate to generate electric fields near MV/cm in suspensions of cells or within tissues (Mankowski, J., Kristiansen, M. (2000) IEEE Trans Plasma Science 28:102-108). Because of their nanosecond duration, the average energy transferred to the cells/tissues by these pulses is theoretically negligible, resulting in electrical effects without accompanying thermal effects.
  • the electric field strength (or electric field intensity) of the nsPEF pulse to be applied to cells is the applied voltage divided by the distance between the electrodes, and is generally at least about 10 kV/cm, but should not exceed the breakdown field of the suspension or tissue which includes the cells.
  • the breakdown field increases with decreasing pulse duration, and can be experimentally determined. Under the conditions commonly employed in the present invention, however, the breakdown field generally does not exceed 500 kV/cm.
  • electric field pulses that have durations of about 300 nanoseconds and typically have electric field strengths greater than 20 kV/cm with rise times of 30 nanoseconds.
  • the pulses should preferably be less than one microsecond, but more than about 100 picoseconds in duration. In one or more aspects of the invention, a pulse duration is about 1 nanosecond to about 300 nanoseconds. The optimum pulse duration will vary depending on the cell type, tissue type, and desired treatment, among other factors. [0038] The number of nsPEF pulses, and the number of any successive treatments to be applied to the tissue, is that sufficient to induce complete regression of the undesired tissue, for example, complete tumor regression. This number may vary based on a variety of factors included the intended effect, the mode of administration of the nsPEFs, and the cells to be treated.
  • the nsPEFs are distinct from electroporation pulses based on their temporal and electrical characteristics, as well as their effects on intact cells and tissues.
  • electroporation pulses and nsPEFs exhibit different electric field strength ⁇ 1-5 kV/cm vs. 10-350 kV/cm); different pulse durations ⁇ 0.1-20 milliseconds vs. 1-300 nanoseconds); different energy densities (joules/cc vs. millijoules/cc) and different power (SOOWvs. 180MW).
  • nsPEFs can be five to six orders of magnitude shorter with electric fields and power several orders of magnitude higher and energy densities considerably lower than electroporation pulses.
  • nsPEFs are exceptional because they are very low energy and extremely high power. Stemming from these differences, as the pulse duration decreases, nsPEFs bypass the plasma membrane and target intracellular structures such as the mitochondria, endoplasmic reticulum, Golgi apparatus, nucleus, or any intracellular store, leaving the plasma membrane intact. These pulses have effects that are unexpectedly different than those of electroporation pulses because, when the pulse duration is short enough and the electric field intensity is high enough, intracellular structures are targeted.
  • the effects of nsPEFs on cells differ depending on the cell type, pulse duration and rise-time, electric field intensity, and/or other factors.
  • nsPEFs and electroporation pulses have different effects on cells.
  • Jurkat cells exposed to classical electroporation pulses e.g.lOO ⁇ s
  • PI immediate propidium iodide
  • nsPEFs have greater plasma membrane effects on smaller cells (e.g. T-cells) than larger ones (e.g. monocytes).
  • nsPEFs Under conditions that are independent of plasma membrane electroporation, nsPEFs have been shown to alter signal transduction mechanisms that determine cell fate. Using nsPEFs, it is possible to trigger apoptosis (Beebe, S. J., et al. (2002), IEEE Trans. Plasma Sci. 30:1 Part 2, 286-292; Beebe, S.J., et al. (2003), FASEBJ (online, June 17, 2003) 10.1096//fj.02-0859fje; Vernier, P.T., et al. (2003), Biochem. Biophys. Res. Comm.
  • nsPEFs induced several well-characterized apoptosis markers including intact plasma membranes, annexin-V- FITC binding, caspase activation, cell shrinkage, cytochrome c release into the cytoplasm, and ultimately, a late secondary necrosis as defined by rupture of the plasma membrane in vitro in the absence of phagocytosis (Beebe et al., 2003).
  • One or more embodiments of the invention are directed to a method of treating melanomas with a second, or multiple, treatments to lead to complete tumor remission.
  • Other embodiments of the invention involve the use of nsPEFs in patients to cause tumor blood flow to stop.
  • the use of nsPEFs in patients cause the inhibition of blood flow to any particular tissue.
  • Example 1 Applying nsPEFs to treat melanomas Materials and Methods
  • Cell tissue culture - Murine melanoma Bl 6-F 10 cells were obtained from ATCC (Manassas, VA) and were stored frozen in liquid nitrogen until needed. They were thawed in a 37°C water bath and then transferred to a culture flask containing DMEM (Dulbecco's Modified Eagles Medium) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals), 4mM L-Glutamine (Cellgro), and 2% Penicillin- Streptomycin solution (Cellgro). The cells were grown in a 5% CO 2 /95% air/100% humidified incubator at 37°C.
  • DMEM Dynabecco's Modified Eagles Medium
  • FBS fetal bovine serum
  • Cellgro 4mM L-Glutamine
  • Penicillin- Streptomycin solution Cellgro
  • In vivo Imaging Melanomas were imaged daily by both transillumination and surface photography at 1.2X magnification and ultrasound images were also taken beginning with mouse 50.
  • Visualsonics Vevo 770 (Visualsonics Inc., Toronto, Canada) was used to image tumors in situ.
  • the power Doppler mode provided blood flow images for each tumor.
  • Histology - Phosphate-buffered formalin (10%) was injected into the loose areolar layer under the skin at the tumor site immediately after euthanizing the mouse and 15 min prior to tumor dissection.
  • the tumor was placed in formalin fixative (minimum 2OX tumor volume) for 24 to 48 h at room temperature.
  • the tumor and surrounding skin were trimmed and both external and internal surfaces were photographed.
  • the fixed tumor was dehydrated through a standard 30%, 50%, 70%, 80%, 90%, 95%, 100% X3 ethanol series, cleared in 100% X 2 xylene, infiltrated at 60 oC in molten paraffin baths X2 (all for 1 h each) and then embedded in paraffin block. Seven ⁇ m thick sections were cut and stained with hematoxylin and eosin.
  • Pulse Generator - A pulse-forming network with an impedance of 75 ⁇ was used. As shown in Figure 1, it consists of 30 pairs of high voltage capacitors and 30 inductors arranged in a Blumlein configuration, and generates a 300 ns long high voltage pulse (J.F.Kolb, S.Kono, K.H.Schoenbach (2006) Bioelectromagnetics. 27(3): 172-87). The pulse was originally triggered by means of a spark gap that was later replaced by a mercury displacement relay controlled by a microcontroller.
  • the voltage across the object was monitored using a high voltage probe (P6015A, Tektronix, Beaverton, CA), and the current was measured by means of a Pearson coil (model 2877, Pearson Electronics Inc., Palo Alto, CA). Current and voltage were recorded simultaneously using a digitizing oscilloscope (TDS3052, Tektronix, Beaverton, OR).
  • Electrodes for electric field application Three types of electrodes were employed; a 5-needle array, a 2-needle array and parallel plates.
  • the 5-needle array ( Figure 2) was made using 30 gauge hypodermic needles (300 ⁇ m diameter) extending 2 mm from a Teflon base. The center needle was the anode and the four surrounding needles spaced 4 mm from the center electrode were connected together forming the cathode.
  • the skin was coated with vegetable oil prior to needle insertion to increase the breakdown field strength along the skin and reduce the likelihood of flashover between needles during the pulsed field application.
  • the parallel plate electrodes ( Figure 6A) were made from stainless steel with diameters of 3-5 mm, depending on the size of the tumor being treated.
  • Electrodes were coated with a 0.5 mm thick layer of conductive agar (IM NaCl in 2% agar) to separate the skin from the electrode.
  • IM NaCl conductive agar
  • each tumor was positioned between two plates with a separation of 0.5-1 mm, while 100 pulses 300 ns in duration and 4-8 kV in amplitude with a rise time of about 30 nanoseconds, were applied at a frequency of 0.5 Hz.
  • Determination ofcaspase activation in vitro Caspase activity was determined in vitro from melanoma tumor extracts after exposure to nsPEF. Melanomas were dissected out of the mouse and frozen in liquid nitrogen.
  • Extracts were prepared from thawed tissue homogenates and assayed for caspase activity using the fluorogenic substrate Ac-DEVD- AFC (Alexis Biochemicals, San Diego, CA) as previously described (L.K.Parvathenani, E.S.Buescher, E.Chacon-Cruz, SJ.Beebe (1998) J.Biol.Chem. 273:6736-6743).
  • This peptide sequence is based on the PARP cleavage site, Asp216, for caspases 1, 3, 4 and 7 that exhibits enhanced fluorescence upon cleavage.
  • the electric field was applied using two different electrode configurations.
  • the first was a 5-needle electrode array (Figure 2A) in which the needles penetrated about 2 mm into the mouse skin.
  • the central needle was placed in the center of the melanoma to be treated and the outer 4 needles were outside of the boundary edges of the melanoma.
  • This electrode array exhibits a sharply non-uniform field with field lines parallel to the surface of the skin and strongest near the center electrode (Figure 2B).
  • Figure 3 H-M When the needle array is inserted into a melanoma for a couple of minutes and removed, the melanoma continues to grow normally (Figure 3 H-M). However, if 100 pulses (8 kV, 300 ns.
  • This tumor response is dependent on both field strength and pulse number. If the field strength is cut in half by using a 4 kV pulse (average field of 10 kV/cm), there is no significant difference between the growth rates of treated and control tumors (Figure 4A). This holds true for the application of both 10 and 100 pulses ( Figure 4B). The pulse number dependence is more evident for the 8 kV pulses (20 kV/cm field) where the response is stronger for 100 pulses than it is for 10 ( Figure 4 C, D) and even stronger when two treatments of 100 pulses are given (Figure 4E). Under this latter condition, the tumors shrink by about 75% within 8 days.
  • the second electrode configuration used involved placing the tumor between two parallel plates (Figure 6A).
  • the electric field between two parallel plates is uniform except at the edges, so that all cells between the plates will be exposed to the same field strength.
  • These electrodes were used when treating 48 mice by lifting a fold of skin containing the melanoma away from the mouse and placing it between the electrodes in such a way that the entire tumor was positioned between the plates.
  • the distance between the plates was typically 0.5-1 mm, depending on tumor thickness. Based on our previous results with needle electrodes, a field strength of 40 kV/cm was employed and the typical response to nanosecond pulses with this electrode configuration is illustrated in Figure 5.
  • Untreated tumor cells exhibited lightly staining pleomorphic nuclei and abundant cytoplasm containing finely dispersed melanin granules (Figure 9).
  • Treated tumors exhibited densely staining, shrunken nuclei and dyshesion of individual cells with coarse intracellular melanin granules as well as aggregated extracellular melanin granules in the widened interstitial spaces.
  • the tumor cell nuclei shrink by 54% within a few minutes after pulsing and by 68% within three hours. No further nuclear shrinkage occurred during the subsequent two weeks as the tumor decreased in size by 90% (Figure 9E). Some of the tumor nuclei elongate along the electric field axis but this is not always observed.
  • the nuclear pyknosis that follows pulse application occurs faster than any previously observed pyknotic response (S.MAlbarenque, K.Doi (2005) Exp.Mol.Pathol. 78:144-149) and may result from either electrodeformation [18] or the direct electric field interaction with cytoskeletal elements associated with the cell's nuclear lamina to generate the nuclear elongation and shrinking (P.K.Wong, W.Tan, C.M.Ho (2005) J.Biomech.
  • nsPEF stimulate murine melanomas to self-destruct by triggering rapid pyknosis and reducing blood flow without significant increases in caspase activity.
  • a reduction in blood flow to tumors has also been observed following electrochemotherapy but does not occur until 24 h after treatment when the bleomycin entry had destroyed the , endothelial cells (G.Sersa, M.Cemazar, C.S.Parkins, D.J.Chaplin (1999) Eur.J.Cancer 35:672-677).
  • nsPEF requires no drugs to achieve this dramatic reduction in tumor blood flow. This cellular response to a new nanosecond time domain of pulsed electric field application is both novel and deadly.
  • NsPEF affects the tumor without disrupting the dermis so that scarring is less likely. NsPEF should also be effective on other tumor types located deeper in the body where a catheter electrode is guided to the tumor. This highly localized and drug-free physical technique offers a promising new therapy for tumor treatment.
  • mice In contrast, 11 of the 14 controls had to be euthanized when their tumors grew to 1.3 cm as specified in our protocol. Three of the controls stopped growing prior to reaching this size and are still alive. These mice were six months old when the B 16 melanoma cells were injected and their immune response may be strong enough to keep the melanomas under control in these three mice. At 120 days since the first treatment for 9 of the experimental mice, and 90 days since the first treatment for 4 of them, these mice remained tumor-free.
  • UV-induced melanomas An important question involves the response of a skin tumor that has arisen from native epidermal cells rather than carcinoma cells that have been injected into the animal. Preliminary studies show that two transgenic mice with UV-induced melanomas on their backs have responded well to a treatment of 300 pulses, 300 ns, 40 kV/cm. Obtaining transillumination data was not possible due to the dark pigmentation of these mice. However, both ultrasound and surface images exhibit the rapid shrinkage of these melanomas (Figure 12).
  • a melanoma tumor where two-needles were placed sequentially along the tumor has caused the tumor to shrink considerably in a 24 hour period as shown in Figure 13.
  • the advantage of a two- or more-unit needle system in a linear array, rather than a coaxial array, is the fact that the needles do not need to be inserted directly into the tumor, and consequently, possible contamination and/or metastasis is avoided.

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Abstract

L'invention concerne des méthodes pour une nouvelle thérapie sans médicament pour traiter des tumeurs solides de la peau par l'application de champs électriques pulsés en nanosecondes (nsPEF). Dans un mode de réalisation de l'invention, les cellules sont des cellules de mélanome, et les nsPEF appliqués pénètrent à l'intérieur de cellules tumorales et causent un rétrécissement rapide des noyaux des cellules tumorales et l'arrêt du flux sanguin tumoral. Cette nouvelle technique permet de viser des cellules tumorales très localisées avec seulement des effets mineurs sur la peau sus-jascente.
PCT/US2007/004844 2006-02-24 2007-02-26 Des champs électriques pulsés en nanoseconde causent l'autodestruction de mélanomes Ceased WO2007100727A2 (fr)

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US12/280,280 US9168373B2 (en) 2006-02-24 2007-02-26 Nanosecond pulsed electric fields cause melanomas to self-destruct
JP2008556458A JP5329978B2 (ja) 2006-02-24 2007-02-26 メラノーマの自己破壊を引き起こすナノ秒パルス電界
AU2007221182A AU2007221182B2 (en) 2006-02-24 2007-02-26 Nanosecond pulsed electric fields cause melanomas to self-destruct
CA2643210A CA2643210C (fr) 2006-02-24 2007-02-26 Des champs electriques pulses en nanoseconde causent l'autodestruction de melanomes
US14/869,286 US9943684B2 (en) 2006-02-24 2015-09-29 Nanosecond pulsed electric fields cause melanomas to self-destruct
US15/922,758 US10905874B2 (en) 2006-02-24 2018-03-15 Nanosecond pulsed electric fields cause melanomas to self-destruct

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US20180200510A1 (en) 2018-07-19
US20110092973A1 (en) 2011-04-21
CA2643210A1 (fr) 2007-09-07
AU2007221182A2 (en) 2008-10-02
WO2007100727A3 (fr) 2008-02-21
US10905874B2 (en) 2021-02-02
US9168373B2 (en) 2015-10-27
JP2009532077A (ja) 2009-09-10
AU2007221182B2 (en) 2011-11-03
CA2643210C (fr) 2018-05-01
AU2007221182A1 (en) 2007-09-07

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